The application of analytical electron microscopy in the localization of individual LDL-binding sites on cell surfaces.
Immuno-cytochemical characterization of human Low-Density Lipoproteins (LDL) is performed by the reaction between colloidal gold particles, being saturated with rabbit-anti-human apoprotein-B, and human LDL-particles, attached to binding-sites on cultured fibroblasts cell membranes. In conventional transmission electronmicrographs (CTEM) the 5-10 nm colloidal-gold particles are observed by contrast at the plasma membranes, although the LDL-particles themselves remained uncontrasted by the classical post-fixation procedures. Post-fixation procedures using the OsO4 plus K4Fe (CN)6 combination did not improve the LDL-particle contrast sufficiently. Application of both glutaraldehyde fixation plus tannic acid and post-fixation with the OsO4 plus K4Fe (CN)6 combination enhanced the contrast sufficiently to visualize single particles on the fibroblasts outer cell surface. The presence of colloidal-gold particles attached to some of these contrasted structures on the plasma membranes characterize the particles as LDL. However, not all particles visualized on the plasma membrane do contain the attached gold particles. The presence of the gold is shown by X-ray microanalysis of the attached contrasted particles. In scanning electron microscopic images (SEM), the total surface of the fibroblasts was covered with numerous small particles. Several particles were identified by the applied X-ray microanalysis as LDL-particles by their gold content, however, others failed to do so.
D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D005292:Ferricyanides / Q000494:pharmacology; D005347:Fibroblasts / Q000648:ultrastructure; D005976:Glutaral / Q000494:pharmacology; D006046:Gold / Q000494:pharmacology; D006801:Humans; D047348:Hydrolyzable Tannins / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000378:metabolism*; D008854:Microscopy, Electron; D009993:Osmium Tetroxide / Q000494:pharmacology; D011817:Rabbits
D005292:Ferricyanides; D047348:Hydrolyzable Tannins; D008077:Lipoproteins, LDL; D006046:Gold; D009993:Osmium Tetroxide; D005976:Glutaral